Di-san junyi daxue xuebao (Jul 2019)
MiR-214 regulates oxidative stress in H9c2 myocardial cells by targeted inhibition of CaMKⅡ
Abstract
Objective To explore the regulative effect of miR-214 on oxidative stress injury induced by hypoxia reoxygenation (H/R) in H9c2 myocardial cells though targeted inhibition of CaMKⅡ. Methods Rat myocardial H9c2 cells were randomly divided into 4 groups, ①control group: H9c2 cells without any treatment; ② H/R group: the myocardial cells were cultured in hypoxia for 24 h and then reoxygenation for 6 h; ③ mimics+H/R group: the myocardial cells were transfected with miR-214 and then cultured with H/R as above; ④ mimics negative control (MNC)+H/R group: the myocardial cells were transfected with miR-214's mimics negative control and then cultured with H/R as above. CCK-8 assay was used to detect the viability of cells. qPCR was adopted to detect the expression of miR-214. Flow cytometry, TUNEL assay, and SOD or MDA detect kits were used to determine the reactive oxygen species (ROS) levels and cell apoptosis. The association of miR-214 with CaMKⅡ was examined by double-luciferase reporter assay, and their changes at mRNA and protein levels were detected by qPCR and Western blotting. After H9c2 cells were transfected with recombinant adenovirus carrying CaMKⅡ gene or the control adenovirus, the regulative effect of miR-214 on oxidative stress of cells was examined by the above methods. Results CCK-8 assay showed that compared with normal group, the cell viability of H/R group was significantly decreased (P < 0.05), while miR-214 overexpression could obviously increase the cell viability (P < 0.05). qPCR results showed that the expression level of miR-214 was markedly downregulated in the H/R group than the control group, while notable increase of miR-214 was observed in the miR-214 mimics group (P < 0.05). Compared with the normal group, the ROS production, MDA level and TUNEL positive cells were obviously increased, while the SOD level was significantly decreased in H/R group (P < 0.05), but opposite trends were observed when the HR group compared to and the miR-214 mimics group (P < 0.05). TargetScan server indicated that there was binding site of miR-214 and CaMKⅡ. The dual luciferase reporter assay also confirmed that miR-214 could bind to CaMKⅡ in H9c2 cells. In addition, qPCR and Western blot results showed that H/R treatment increased the expression of CaMKⅡ at mRNA and protein levels, but miR-214 significantly inhibited the enhanced expression induced by H/R. Overexpression of CaMKⅡ in H9c2 cells resulted in partially blocked effects of miR-214 on oxidative stress and apoptosis induced by H/R treatment. Conclusion Overexpression of miR-214 can regulate oxidative stress and apoptosis of H9c2 cells by inhibiting CaMKⅡ at the transcription level.
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